1Department of Immunology, School of Medicine, Universidad Autónoma de San Luis Potosí, San Luis Potosí, Mexico.

Abstract

Human papillomavirus (HPV) is able to inhibit the secretion of gamma interferon (IFN-γ) and the expression of some immune innate cell receptors. Immunoglobulin-like transcript 2 (ILT2) is a regulatory receptor that seems to participate in the pathogenesis of viral infections. We have studied the expression and function of ILT2 and the expression of other NK cell receptors in 23 healthy women before and after immunization with the quadrivalent HPV (type 6/11/16/18) vaccine (Gardasil). Receptor expression was analyzed by flow cytometry in freshly isolated peripheral blood mononuclear cells as well as after in vitro stimulation with the quadrivalent HPV (type 6/11/16/18) vaccine. In addition, the regulatory function of ILT2 on cell proliferation and IFN-γ production was analyzed. We found a significant increase in the expression of ILT2 by NK and CD3(+) CD56(+) lymphocytes and monocytes after quadrivalent HPV (type 6/11/16/18) vaccine immunization. In addition, the in vitro stimulation with the quadrivalent HPV (type 6/11/16/18) vaccine also increased the proportion of CD3(-) CD56(+) ILT2(+) NK cells. Although the inhibitory function of ILT2 on cell proliferation was enhanced after HPV immunization, the in vitro engagement of this receptor did not affect the synthesis of IFN-γ induced by HPV. Finally, a significant increase in the expression of NKG2D, NKp30, and NKp46 by NK and CD3(+) CD56(+) lymphocytes was detected after quadrivalent HPV (type 6/11/16/18) vaccine immunization. Our data indicate that HPV immunization is associated with significant changes in the expression and function of different innate immune receptors, including ILT2, which may participate in the protective effect of HPV vaccines.

Expression of ILT2 after HPV immunization. The percentage of ILT2+ cells in different leukocyte subsets of PBMCs was determined by flow cytometry in 23 healthy volunteers before (T0) and 15 days after the first (T1, day 15) and third (T2, day 195) quadrivalent HPV (type 6/11/16/18) vaccine doses. (A) Flow cytometry histograms of ILT2 expression by CD8+ lymphocytes at T0 (upper panel) and T2 (lower panel) of quadrivalent HPV (type 6/11/16/18) vaccine immunization. Data from a representative individual are shown. The thick lines correspond to cells stained with the anti-ILT2 MAb, and thin lines correspond to cells incubated with an isotype-matched MAb. (B to G) Expression levels of ILT2 by the indicated subsets of PBMCs are shown as the arithmetic mean and SD of the percentage of positive cells in nonstimulated cells and cells stimulated in vitro for 72 h with HPV (a mixture of 10 ng/ml of L1 protein of HPV-6 and -18 and 20 ng/ml of HPV-11 and -16 from the Gardasil vaccine). *, P < 0.05; **, P < 0.01; ***, P < 0.001 (two-way repeated measures analysis of variance).

Analysis of expression of NK cell receptors after HPV immunization. (A to F) PBMCs from 23 healthy volunteers were obtained before (T0) and 15 days after the first (T1, day 15) and third (T2, day 195) quadrivalent HPV (type 6/11/16/18) vaccine doses. These cells were immunostained with anti-CD3, -CD56, and the indicated MAbs, and the percentage of positive cells was analyzed by flow cytometry. Data correspond to the expression of the indicated antigens in CD3− CD56+ or CD3+ CD56+ cells. (G to I) MFI of expression of the indicated receptors by NK cells (CD3− CD56+) in PBMCs from the same individual whose data are reflected in panels A to F. Data correspond to the arithmetic mean and SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (one-way repeated measures analysis of variance).

Regulatory function of ILT2 after HPV immunization. (A) PBMCs from 23 healthy volunteers were obtained before (T0) and 15 days after the first (T1, day 15) and third (T2, day 195) quadrivalent HPV (type 6/11/16/18) vaccine doses. Cells were loaded with CFSE and incubated for 72 h in the presence of the quadrivalent HPV (type 6/11/16/18) vaccine and with the addition (empty bars) or not (black bars) of the agonistic HP-F1 anti-ILT2 or an isotype-matched MAb (gray bars). Then, the percentage of divided cells was determined by flow cytometry analysis, as stated in Materials and Methods. Data correspond to the arithmetic mean and SD. *, P < 0.05, two-way repeated measures analysis of variance. (B) PBMCs from the same individuals whose data are represented in panel A were incubated for 72 h in the presence of quadrivalent HPV (type 6/11/16/18) vaccine and with the addition (empty bars) or not (black bars) of the agonistic HP-F1 anti-ILT2 or an isotype-matched MAb (gray bars). Then, cell nuclei were stained with propidium iodide and analyzed for DNA content by flow cytometry. Data correspond to the arithmetic mean and SD. *, P < 0.05, two-way repeated measures analysis of variance.

Analysis of IFN-γ release after HPV immunization. PBMCs from 23 healthy volunteers were obtained before (T0) and 15 days after the first (T1, day 15) and third (T2, day 195) quadrivalent HPV (type 6/11/16/18) vaccine doses. Cells were stimulated (empty bars) or not (black bars) with the quadrivalent HPV (type 6/11/16/18) vaccine for 72 h, in the presence or absence of an agonistic anti-ILT2 antibody, and the number of IFN-γ-producing cells was analyzed by an ELISPOT assay. Data correspond to the arithmetic mean and SD. ***, P < 0.001 (two-way analysis of variance); ns, nonsignificant.